Single molecule sequencing of captured nucleic acids

ABSTRACT

The invention provides methods and devices for detecting, enumerating or identifying target nucleic acid molecules using immobilized capture probes and single molecule sequencing techniques.

TECHNICAL FIELD OF THE INVENTION

This invention relates to methods and compositions for detecting,enumerating, and identifying nucleic acids using capture probes tocapture and purify target nucleic acid in combination with singlemolecule sequencing of the captured or purified nucleic acid molecules.

BACKGROUND OF THE INVENTION

Knowledge of the human genome has given rise to inquiry into individualdifferences, as well as differences within an individual, as the basisfor differences in biological function and dysfunction. Differences assmall as single nucleotide polymorphisms (SNPs) or combinations of SNPscan lead to phenotypic differences, and detection of combinations ofSNPs can predict the likelihood that an individual will get a specificdisease or how an individual will respond to treatment.

For example, most cancers develop from a series of genomic changes, somesubtle and some significant, that occur in a small subpopulation ofcells. Knowledge of the sequence variations that lead to cancer willlead to an understanding of the etiology of the disease, as well as waysto treat and/or prevent it. An essential first step in understandinggenomic complexity is the ability to perform high-resolution sequencing.Therefore, a true understanding of the complexities in either normal orabnormal function will require specific sequence information from largenumbers of target nucleic acid molecules.

Bulk sequencing techniques are often not useful for the identificationof subtle or rare nucleotide changes due to the many cloning,amplification and electrophoresis steps that complicate the process ofgaining useful information regarding individual nucleotides. The abilityto sequence and gain information from single molecules obtained from anindividual patient is the next milestone for genomic sequencing.However, effective diagnosis and management of important diseasesthrough single molecule sequencing is impeded by lack of cost-effectivetools and methods for screening individual molecules.

There have been many proposals to develop new sequencing technologiesbased on single-molecule measurements, generally either by observing theinteraction of particular proteins with DNA or by using ultra highresolution scanned probe microscopy. See, e.g., Rigler, et al.,Biotech., 86 (3):161 (2001); Goodwin, P. M., et al., Nucleosides &Nucleotides, 16 (5-6):543-550 (1997); Howorka, S., et al., NatureBiotech., 19 (7):636-639 (2001); Meller, A., et al., Proc. Natl. Acad.,97 (3):1079-1084 (2000); Driscoll, R. J., et al., Nature, 346(6281):294-296 (1990). A recent technique employs optical detection in asequencing-by-synthesis reaction at the single molecule level.Braslaysky, et al., PNAS, 100: 3960-3964 (2003). The present inventionprovides improvements in sequencing, especially single moleculesequencing.

SUMMARY OF THE INVENTION

The invention provides methods for sequencing nucleic acids. Accordingto the invention, sequencing is facilitated by capture of target nucleicacids to be sequenced prior to sequencing. In a preferred embodiment,target nucleic acids are sequenced at the single molecule level,resulting in sample-specific, high-throughput sequence information.Thus, according to one aspect of the invention, sequence-specificcapture probes are used to isolate target nucleic acids of interest. Thetarget population may be composed of same-sequence nucleic acids or maybe a population of mixed-sequence nucleic acids. The isolated targetnucleic acids are then sequenced. In one embodiment, sequencing isperformed directly on captured nucleic acids, using the capture probesas primers. Alternatively, target/probe duplex may be melted and thetarget nucleic acids may then be hybridized to primers fortemplate-dependent sequencing.

In a preferred embodiment, single molecule sequencing is conducted inorder to provide high-resolution, high-throughput sequence information.Template-dependent single-molecule sequencing-by-synthesis is conductedusing optically-labeled nucleotides for addition to the primer or probe.Either the target (template) or the primer/probe, or both are attachedto a surface that is designed to enhance optical signal detection. Aparticularly-preferred surface is an epoxide surface coated onto glassor fused silica. Nucleic acids are easily attached to epoxide or epoxidederivatives. A preferred method is direct amine attachment. Nucleicacids can be purchased with a 5′ or 3′ amine, or terminal transferasecan be used to introduce a terminal amine for attachment to the epoxidering. Alternatively, epoxide surfaces can be derivatized for nucleicacid attachment. For example, the surface can incorporate streptavidin,which binds to biotinylated nucleic acids. Either the target,primer/probe, or both can be biotinylated using known methods.Alternative surfaces include polyelectrolyte multilayers as described inBraslavasky, et al., PNAS 100: 3960-64 (2003), incorporated by referenceherein. Essentially, any surface that has reduced native fluorescenceand is amenable to attachment of oligonucleotides is useful for theinvention.

Single molecule sequence is advantageously performed usingoptically-detectable labels. Especially preferred are fluorescentlabels, including fluorescein, rhodamine, derivatized rhodamine dyes,such as TAMRA, phosphor, polymethadine dye, fluorescent phosphoramidite,texas red, green fluorescent protein, acridine, cyanine, cyanine 5 dye,cyanine 3 dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid(EDANS), BODIPY, 120 ALEXA, or a derivative or modification of any ofthe foregoing.

The capture step prior to sequencing may be any suitable hybrid capturemethod. For example, capture can occur in solution, on beads(polystyrene beads), in a column (such as a chromatography column), in agel (such as a polyacrylamide gel), or directly on the surface to beused for sequencing. An array of support-bound capture oligos can beused to hybridize specifically to a target sequence. Additionally,chromatography-based capture techniques are useful. For example, ionexchange chromatography, HPLC, gas chromatography, and gel-basedchromatography all are useful. In one embodiment, gel-based capture isused in order to achieve sequence-specific capture. Using this method,multiple different sequences are captured simultaneously usingimmobilized probes in the gel. The target sequences are isolated byremoving portions of the gel containing them and eluting target from thegel portions for sequencing.

In an alternative embodiment, the target nucleic acid molecule eitherincludes, or is modified to include, an adaptor sequence (such as apolyadenylation region) that is complementary to a portion of a captureprobe in order to aid in the capture of the target. A preferredembodiment comprises an immobilized capture probe having a sequence thathybridizes (e.g., is complementary to) with the adaptor sequence.Methods of the invention are conducted by contacting capture probes witha sample comprising target nucleic acid molecules under conditionssuitable for specific hybridization between the target nucleic acidmolecule and immobilized capture probe, thereby forming target/captureprobe duplex. A wash step removes debris and unhybridized nucleic acidin the sample. In one embodiment, target nucleic acid is sequenced usingthe capture probe as a primer for template-dependentsequencing-by-synthesis. In another embodiment, the target/capture probeduplex is melted to release target nucleic acid. The resulting purifiedtarget population is sequenced as described below.

If target nucleic acid is melted off the capture probe, the targets areeither attached to a surface for sequencing or hybridized to a primerthat has been attached to the surface. Surface attachment ofoligonucleotides for sequencing can be direct or indirect. For example,nucleic acids are attached to an epoxide surface via a direct aminelinkage as described below. Alternatively, the surface is prepared witha binding partner, the opposite of which is attached to the nucleicacid. For example, the surface can be streptavidinated and biotinylatednucleic acids can be used to form an attachment at the surface. Otherbinding pairs (e.g., antibody/antigen, such asdigoxigenen/anti-digoxigenen and dinitorphenol/anti-dinitrophenol) canalso be used.

In a preferred embodiment, the invention contemplates opticalsequencing. Preferably, template-dependent sequencing-by-synthesis isconducted using optically-detectable labels. Optimal labels includefluorescent labels as described in detail below. The surface is preparedto minimize background for optical detection of incorporatednucleotides. Primer/template duplex attached to the surface is exposedto labeled nucleoside triphosphates in the presence of a suitablenucleotide polymerizing enzyme, under conditions suitable for the enzymeto add at least one nucleotide to the primer in template-dependentmanner, and the added nucleotide is detected, thereby identifying atleast one nucleotide in at least one attached target nucleic acidmolecule.

As will be appreciated by one skilled in the art, individual features ofthe invention may be used separately or in any combination. A detaileddescription of embodiments of the invention is provided below. Otherembodiments of the invention are apparent upon review of the detaileddescription that follows.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for sequencing nucleic acids. Ina preferred embodiment, the invention comprises the use of singlemolecule sequencing of hybrid-captured nucleic acids. Methods of theinvention are highly parallel and are amenable to multiplexing. As aresult, biological samples can be rapidly analyzed over a broad dynamicrange.

Methods of the invention comprise hybrid-capturing target nucleic acidand sequencing the captured targets. Captured target nucleic acids canbe sequenced directly using the capture probe as a primer fortemplate-dependent synthesis. Thus, capture probes are bound to asurface and exposed to sample nucleic acids. Complementary binding oftarget nucleic acid and probes occurs and the remaining nucleic acid andother sample contents are washed from the surface. Sequencing then takesplace essentially as described below. In one alternative, target nucleicacids are isolated by contacting a sample containing target nucleic acidmolecules with immobilized capture probes capable of hybridizing totarget nucleic acid. Target nucleic acid The surface is washed to removenon-target nucleic acids and other debris, and the targets are thenmelted off the target/capture probe duplex. After an optional wash, thetargets are bound to a surface for sequencing essentially as describedbelow. Target nucleic acid molecules preferably are analyzed usingsingle molecule sequencing techniques described below. In a preferredembodiment, target nucleic acid is modified by addition of apolynucleotide sequence that specifically hybridizes to a complementaryportion of a primer for nucleic acid synthesis. For example, targets canbe polyadenylated and captured using poly-dT probes/primers forsequencing.

Target Nucleic Acid Molecules

Target nucleic acids include deoxyribonucleic acid (DNA) and/orribonucleic acid (RNA). Target nucleic acids can be isolated or can becaptured in situ. In a preferred alternative, target nucleic acid isisolated from a biological sample containing a variety of othercomponents, such as proteins, lipids and non-target nucleic acids.Target nucleic acid molecules can be obtained from any cellularmaterial, obtained from an animal, plant, bacterium, virus, fungus, orany other cellular organism. Target nucleic acids may be obtaineddirectly from an organism or from a biological sample obtained from anorganism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid,saliva, sputum, stool and tissue. Any tissue or body fluid specimen maybe used as a source for nucleic acid for use in the invention. Nucleicacid molecules may also be isolated from cultured cells, such as aprimary cell culture or a cell line. The cells from which target nucleicacids are obtained can be infected with a virus or other intracellularpathogen.

A sample can also be total RNA extracted from a biological specimen, acDNA library, or genomic DNA. Nucleic acid typically is fragmented toproduce suitable fragments for capture and/or purification and analysis.In one embodiment, nucleic acid from a biological sample is fragmentedby sonication. Test samples can be obtained as described in U.S. PatentApplication 2002/0190663 A1, published Oct. 9, 2003, the teachings ofwhich are incorporated herein in their entirety. Generally, nucleic acidcan be extracted from a biological sample by a variety of techniquessuch as those described by Maniatis, et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982).Generally, target nucleic acid molecules can be from about 5 bases toabout 20 kb. Nucleic acid molecules may be single-stranded,double-stranded, or double-stranded with single-stranded regions (forexample, stem- and loop-structures).

A biological sample as described herein may be homogenized orfractionated in the presence of a detergent or surfactant. Theconcentration of the detergent in the buffer may be about 0.05% to about10.0%. The concentration of the detergent can be up to an amount wherethe detergent remains soluble in the solution. In a preferredembodiment, the concentration of the detergent is between 0.1% to about2%. The detergent, particularly a mild one that is nondenaturing, canact to solubilize the sample. Detergents may be ionic or nonionic.Examples of nonionic detergents include triton, such as the Triton® Xseries (Triton® X-100 t-Oct-C₆H₄—(OCH₂—CH₂)_(x)OH, x=9-10, Triton®X-100R, Triton® X-114 x=7-8), octyl glucoside, polyoxyethylene(9)dodecylether, digitonin, IGEPAL® CA630 octylphenyl polyethylene glycol,n-octyl-beta-D-glucopyranoside (betaOG), n-dodecyl-beta, Tween® 20polyethylene glycol sorbitan monolaurate, Tween® 80 polyethylene glycolsorbitan monooleate, polidocanol, n-dodecyl beta-D-maltoside (DDM),NP-40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycoln-dodecyl monoether), hexaethyleneglycol mono-n-tetradecyl ether(C14E06), octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG),Emulgen, and polyoxyethylene 10 lauryl ether (C12E10). Examples of ionicdetergents (anionic or cationic) include deoxycholate, sodium dodecylsulfate (SDS), N-lauroylsarcosine, and cetyltrimethylammoniumbromide(CTAB). A zwitterionic reagent may also be used in the purificationschemes of the present invention, such as Chaps, zwitterion 3-14, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. It iscontemplated also that urea may be added with or without anotherdetergent or surfactant.

Lysis or homogenization solutions may further contain other agents, suchas reducing agents. Examples of such reducing agents includedithiothreitol (DTT), β-mercaptoethanol, DTE, GSH, cysteine, cysteamine,tricarboxyethyl phosphine (TCEP), or salts of sulfurous acid.

Preferred buffer concentration is from about 5 mM to about 500 mM insolution or in solution with the sample. The buffer concentration in thelysing solution can be between about 10 mM and 300 mM.

Subsequent to exposure to a homogenization solution, samples may befurther homogenized by mechanical means. Mechanical blenders,rotor-stator homogenizers, or shear-type homogenizers may be employed.Alternatively, the tissue can be homogenized in the lysis solution, andthe tissue remains separated by settling, centrifugation, or filtration.These remains could then be treated with homogenization solution andextraction conditions as described above.

Target nucleic acid molecules can include or be modified to include anadaptor sequence that is complementary to the immobilized capture probe.The adaptor sequence and immobilized capture probe are chosen such thatthe target nucleic acid molecule comprising the adaptor sequence canhybridize to the immobilized capture probe. Typically, the adaptorsequence is a homopolymer, such as oligo(dA), and the correspondingimmobilized capture probe includes an oligo(dT) sequence. The adaptorsequence may be endogenously contained within the target nucleic acidmolecule, for example, the adaptor sequence can be a sequence ofinterest in the target nucleic acid molecule. Alternatively, the adaptorsequence can comprise vector sequence.

In one embodiment, a universal probe is used as the immobilized captureprobe and the target nucleic acid molecules are modified with an adaptorcomprising a sequence complementary to the universal probe, therebyallowing the modified nucleic acid molecules to hybridize to theimmobilized probe.

The adaptor sequence and complementary sequence within the immobilizedcapture probe are of a length suitable for hybridizing the targetnucleic acid molecule to the immobilized capture probe to therebycapture target nucleic acid molecules to form target probe/duplexes. Thetarget probe/duplexes are also sufficiently stable to permit optionalwashing of the duplexes to remove any remaining components of thebiological sample, including non-target nucleic acids. The sequence ofthe adaptor and the complementary sequence of the immobilized captureprobe can be about 10 to about 100, and preferably 50, nucleotides inlength. The adaptor sequence and complementary immobilized capturesequence can be of the same length or of different lengths. It isroutine in the art to adjust probe length and/or oligonucleotide lengthto optimize hybridization.

The adaptor sequence can be attached to the nucleic acid molecules withan enzyme. The enzyme can be a ligase or a polymerase. The ligase can bean enzyme capable of ligating an oligonucleotide (RNA or DNA) to thenucleic acid molecules. Suitable ligases include, for example, T4 DNAligase and T4 RNA ligase (such ligases are available commercially, fromNew England BioLabs (on the World Wide Web at NEB.com). In a preferredembodiment, the target nucleic acid molecules are dephoyshorylatedbefore ligating the adaptors. Methods for using ligases are well knownin the art.

The polymerase can be any enzyme capable of adding nucleotides to thetarget nucleic acid molecules. The polymerase can be, for example,poly(A) polymerase, including yeast poly(A) polymerase, commerciallyavailable from USB (on the World Wide Web at USBweb.com), terminaldeoxyribonucleotidyl transferase (TdT), and the like. The polymerasescan be used according to the manufacturer's instructions.

Purifying Target Nucleic Acid Molecules

As described herein, target nucleic acid molecules can be purified bycontacting a sample containing the target molecules with one or moreclasses of immobilized capture probes. Where more than one targetmolecule is to be purified, the different target molecules can have thesame or different adaptor sequences. A given class of immobilizedcapture probe is selected to hybridize to target nucleic acid moleculescontaining the corresponding adaptor sequence, under conditions suitablefor the target nucleic acid molecule to hybridize to the immobilizedcapture probe, thereby forming target/probe duplexes. A variety ofcapture probes can be used to purify the target nucleic acid molecules.The capture probes typically comprise a nucleic acid with a nucleotidesequence with substantial complementarity to a region of the targetnucleic acid molecule, so that the target nucleic acid molecule canhybridize to the capture probe. Complementarity between target nucleicacid molecules and the capture probes need only be sufficient tospecifically bind the target nucleic acid molecule, and thus, toeffectuate purification of the target nucleic acid molecule from a testsample. Probes suitable for use in the present invention include thoseformed from nucleic acids, such as RNA and/or DNA, nucleic acidanalogues, modified nucleic acids, and chimeric probes of a mixed classcomprising a nucleic acid with another organic component such as peptidenucleic acids. Capture probes can be single stranded or double stranded.Preferably, the length of the capture probe is at least 5 nucleotides,more preferably between about 5 and about 100 nucleotides, but thelength can be up to several thousand nucleotides. Additional probes aredescribed in U.S. Patent application 2002/0119480 A1, published Aug. 29,2002, the teachings of which are incorporated herein in their entirety.

Capture probes can be coupled to agarose, dextrans, cellulose, beads,microparticles, and starch polymers using cyanogen bromide or cyanuricchloride activation. Particularly useful beads and microparticles aredescribed in U.S. Patent Application 2003/0190663 A1, published Oct. 9,2003, the teachings of which are incorporated herein in their entirety.Polymers containing carboxyl groups can be coupled to synthetic captureprobes having primary amine groups using carbodiimide coupling. Polymerscontaining primary amines can be coupled to amine-containing probes withglutaraldehyde or cyanuric chloride. Polymers can also be modified withthiol-reactive groups that can be coupled to thiol-containing probes.Many other suitable methods can be found in the literature (Wong,Chemistry of Protein Conjugation and Cross-Linking, CRC Press, BocaRaton, Fla. 1993). Methods for covalently attaching the capture probesto polymerizable chemical groups are also described in U.S. 2002/0119480A1, the teachings of which are incorporated herein. In addition, asdescribed in U.S. 2002/0119480 A1, nucleic acids can be attached toparticles which themselves can be incorporated into electrophoreticmedia.

To purify target nucleic acid molecules, the test sample can becontacted, for example, with an electrophoretic medium that comprises atleast one immobilized capture probe. An electric field is applied acrossthe electrophoretic medium so that negatively charged molecules canmigrate through the medium. The non-target components of the test samplepass into the electrophoresis buffer, while the target nucleic acidmolecules are captured by hybridizing to the capture probes within theelectrophoretic medium (forming target/probe duplexes). The buffer usedfor the electrophoresis step (containing the non-target components ofthe test sample) can be replaced with fresh buffer and current appliedacross the electrophoretic medium to denature the complex, therebyreleasing the target nucleic acid molecule from the capture probe. Thereleased target nucleic acid molecule can be eluted by applying areversed electric field.

The method of the present invention is suitable for multiplexing. Asdescribed above, a sample can include more than one target nucleic acidmolecule. If adapters are used, the different target nucleic acidmolecules can have the same or different adaptor sequences. In amultiplexing reaction, where the target nucleic acid molecules containdifferent adaptor sequences, the test sample is contacted with two ormore classes of immobilized capture probes as described above. Targetnucleic acid molecules are captured by their respective immobilizedcapture probes. The captured target nucleic acid molecules can bereleased as described above. Other methods for capturing and purifyingtarget nucleic acid molecules are described in U.S. 2002/0119480 A1, theteachings of which are incorporated herein.

Attaching Target Nucleic Acid Molecules To A Surface

In a preferred embodiment, target nucleic acid molecules are attached toa surface and subjected to analysis by single molecules sequencing.Target nucleic acid molecules are attached to the surface such that theyare individually optically resolvable. Substrates for use in theinvention can be two- or three-dimensional and can comprise a planarsurface (e.g., a glass slide) or can be shaped. A substrate can includeglass (e.g., controlled pore glass (CPG)), quartz, plastic (such aspolystyrene (low cross-linked and high cross-linked polystyrene),polycarbonate, polypropylene and poly(methymethacrylate)), acryliccopolymer, polyamide, silicon, metal (e.g., alkanethiolate-derivatizedgold), cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel),polyacrolein, or composites.

Suitable three-dimensional substrates include, for example, spheres,microparticles, beads, membranes, slides, plates, micromachined chips,tubes (e.g., capillary tubes), microwells, microfluidic devices,filters, or any other structure suitable for anchoring a nucleic acid.Substrates can include planar arrays or matrices capable of havingregions that include populations of target nucleic acids or primers.Examples include nucleoside-derivatized CPG and polystyrene slides;derivatized magnetic slides; polystyrene grafted with polyethyleneglycol, and the like.

In one embodiment, a substrate is coated to allow optimum opticalprocessing and nucleic acid attachment. Surfaces for use in theinvention are treated to reduce background. Exemplary coatings includeepoxides, and derivatized epoxides (e.g., with a binding molecule, suchas streptavidin). The surface can also be treated to improve thepositioning of attached nucleic acids (e.g., target nucleic acidmolecules, primers, or target molecule/primer complexes) for analysis.As such, a surface according to the invention can be treated with one ormore charge layers (e.g., a negative charge) to repel a charged molecule(e.g., a negatively charged labeled nucleotide). For example, asubstrate according to the invention can be treated with polyallylaminefollowed by polyacrylic acid to form a polyelectrolyte multilayer. Thecarboxyl groups of the polyacrylic acid layer are negatively charged andthus repel negatively charged labeled nucleotides, improving thepositioning of the label for detection. Coatings or films applied to thesubstrate should be able to withstand subsequent treatment steps (e.g.,photoexposure, boiling, baking, soaking in warm detergent-containingliquids, and the like) without substantial degradation or disassociationfrom the substrate.

Examples of substrate coatings include, vapor phase coatings of3-aminopropyltrimethoxysilane, as applied to glass slide products, forexample, from Molecular Dynamics, Sunnyvale, Calif. In addition,generally, hydrophobic substrate coatings and films aid in the uniformdistribution of hydrophilic molecules on the substrate surfaces.Importantly, in those embodiments of the invention that employ substratecoatings or films, the coatings or films that are substantiallynon-interfering with primer extension and detection steps are preferred.Additionally, it is preferable that any coatings or films applied to thesubstrates either increase target molecule binding to the substrate or,at least, do not substantially impair target binding.

Various methods can be used to anchor or immobilize the target nucleicacid molecule to the surface of the substrate. The immobilization can beachieved through direct or indirect bonding to the surface. The bondingcan be by covalent linkage. See, Joos et al., Analytical Biochemistry247:96-101, 1997; Oroskar et al., Clin. Chem. 42:1547-1555, 1996; andKhandjian, Mole. Bio. Rep. 11:107-115, 1986. A preferred attachment isdirect amine bonding of a terminal nucleotide to an epoxide integratedon the surface. The bonding also can be through non-covalent linkage.For example, biotin-streptavidin (Taylor et al., J. Phys. D. Appl. Phys.24:1443, 1991) and digoxigenin with anti-digoxigenin (Smith et al.,Science 253:1122, 1992) are common tools for anchoring nucleic acids tosurfaces and parallels. Alternatively, the attachment can be achieved byanchoring a hydrophobic chain into a lipid monolayer or bilayer. Othermethods for known in the art for attaching nucleic acid molecules tosupports also can be used.

In a preferred embodiment, surfaces for oligonucleotide attachment arecoated with an epoxide. An epoxide may be deposited by many methodsknown in the art. An epoxy silane surface is preferred. Differentmolecules or combinations of molecules may serve to link the epoxide toa surface. Ideally, a surface will be coated with an even distributionof epoxides prior to introduction of target nucleic acid molecules.Target nucleic acid molecules can be directly or indirectly linked to anepoxide. In a direct attachment embodiment, the epoxide is introduced toa nucleic acid bearing an amine group. In a preferred embodiment,terminal transferase is used to add an amine-terminated nucleotide to anucleic acid to be attached to the surface.

When biotin-streptavidin linkage is used to anchor the nucleic acids,the nucleic acids can be biotinylated, while one surface of thesubstrates can be coated with streptavidin.

Surface density of the nucleic acid molecules can be controlled byadjusting the concentration of the streptavidin applied to the surface.Reagents for biotinylating a surface can be obtained, for example, fromVector Laboratories. Alternatively, biotinylation can be performed withBLCPA: EZ-Link Biotin LC-PEO-Amine (Pierce, Cat. 21347), or any otherknown or convenient method. In some embodiments, labeled streptavidin ofvery low concentration (e.g., in the μM, nM or pM range) is used to coatthe substrate surface prior to anchoring. This can facilitateimmobilization of the nucleic acid with single molecule resolution.

Other examples of linkers include antigen/antibody,digoxigenin/anti-digoxigenin, dinitrophenol, fluorescein, and otherhaptens known in the art. Alternatively, the nucleic acid may containother binding moieties that result in a conformational change of theepoxide ring and result in a direct attachment of the target nucleicacid molecules to the opened epoxide ring.

Alternatively, primers may be immobilized on the surface. A terminus ofone or more primers may be modified to carry a linker moiety fortethering the primers to the surface or may be directly attached to thesurface. Target nucleic acid molecules containing primer complementarysequence are then hybridized to the attached primer. Methods forattaching nucleic acid such as primer to the surface of a substrate aredescribed in detail above.

In order to inhibit non-specific binding of molecules to a surface, thesurface can be treated with a passivating (blocking) agent. Preferredblocking strategies include exposing the surface to a non-detectablemolecule that adheres to the surface or changes the chemical propertiesof the surface such that non-specific binding is reduced. In methods inwhich optically-detectable labels are used, one way to block orpassivate the surface is to expose the surface to unlabeled molecules ofthe same type as those that are labeled. The unlabeled molecules willout-compete labeled molecules for non-specific binding on the surface,thus reducing background due to non-specific label. Other strategiesinvolve treating the surface with phosphate, Tris, a sulfate, or anamine that interacts with the surface to prevent non-specific binding.Non-reactive proteins are also appropriate. In a preferred embodiment, amatrix of blocking reagents is provided on the surface in order toprovide a highly washable, low non-specific background surface. In someembodiments, blocking reagents are chosen to provide electrostaticrepulsion of highly anionic nucleoside triphosphates.

Blocking agents may be introduced or reintroduced at any time during theanalysis. Also, in some embodiments, blocking agents may be used topre-treat the surface prior to exposing the substrate to target nucleicacid molecules or primers. In addition, blocking agents, such as adetergent (e.g., Tris) may be included in some or all wash steps inorder to passivate the surface during incubation periods and/or washes.

Surface charge can be manipulated to achieve ideal conditions duringboth nucleic acid attachment and primer extension. For example, duringthe loading phase where the nucleic acid (target nucleic acid moleculeor primer) is bound to the surface, the salt concentration of thesolution may be increased in order to create a more positive surfacecharge on the substrate to facilitate reaction between the amine portionof the nucleic acid and the epoxide ring. Conversely, after the nucleicacid has been secured to the surface, the salt concentration of thesolution can lowered in order to repel the nucleic acid from the surfaceof the substrate thereby sterically conforming the nucleic acid forannealing and sequence analysis.

In another embodiment, the substrate includes a layer of polyanions andnucleic acid molecules anchored on the layer of polyanions. Accordingly,nucleic acids are positioned to avoid being substantially parallel(e.g., is hindered from lying down on the layer of polyanions.) In someembodiments, the surface of a substrate is pretreated to create asurface chemistry that facilitates nucleic acid attachment andsubsequent annealing and sequence analysis. In some of theseembodiments, the substrate surface is coated with a polyelectrolytemultilayer (PEM). In some cases, biotin can be applied to the PEM,followed by application of streptavidin. The substrate can then be usedto attach biotinylated target nucleic acid molecules.

In some embodiments, multiple layers of alternating positive andnegative charges are used. In the case of incompletely-charged surfaces,multiple-layer deposition tends to increase surface charge to awell-defined and stable level. For example, surfaces can be coated witha PEM for attachment of primers via light-directed spatial attachment.Alternatively, target nucleic acid molecules can be attached to aPEM-coated surface chemically. PEM formation has been described inDecher et al. (Thin Solid Films, 210:831-835, 1992), the teachings ofwhich are incorporated herein. PEM formation proceeds by the sequentialaddition of polycations and polyanions, which are polymers with manypositive or negative charges, respectively. Upon addition of apolycation to a negatively-charged surface, the polycation deposits onthe surface, forming a thin polymer layer and reversing the surfacecharge. Similarly, a polyanion deposited on a positively charged surfaceforms a thin layer of polymer and leaves a negatively charged surface.Alternating exposure to poly(+) and poly(−) generates a polyelectrolytemultilayer structure with a surface charge determined by the lastpolyelectrolyte added. This can produce a strongly-negatively-chargedsurface, repelling the negatively-charged nucleotides. Detailedprocedures for coating a substrate with PEM for immobilizing nucleicacid are described in U.S. patent application Ser. No. 11/137,928, fieldMay 25, 2005.

In another aspect of the invention, the substrate may be prepared by,for example, coating with a chemical that increases or decreaseshydrophobicity or coating with a chemical that allows covalent linkageof the primers. Some chemical coatings may both alter the hydrophobicityand allow covalent linkage. Hydrophobicity on a solid substrate mayreadily be increased by silane treatment or other treatments known inthe art. Linker molecules adhere to the surface and comprise afunctional moiety that reacts with biomolecules. Many such linkers arereadily available and known in the art. For example, substrates orsupports are modified with photolabile-protected hydroxyl groups, alkoxyor aliphatic derivatized hydroxyl groups, or other chemicals. Apreferred coating that both decreases hydrophobicity and provideslinkers is poly(ethyleneimine).

Methods of the invention also optionally include a surface drying step.In some embodiments, the surface is exposed to a drying agent prior to,during and/or after a chemical reaction, such as a nucleotideincorporation step. Examples of preferred drying agents include, withoutlimitation, phosphate buffer, an alcohol (such as, for example, EtOH),air and/or N₂.

Analyzing Attached Target Nucleic Acid Molecules

As described herein, attached target nucleic acid molecules are analyzedby single molecule sequencing. At least one nucleotide is identified inat least one attached target nucleic acid molecule. Target molecules arehybridized to a primer to form nucleic acid target molecule/primerduplex on a surface. As described above, either the target nucleic acidmolecule or the primer, or both, is/are attached to the surface.Thereafter, template-dependent primer extension is conducted to identifyat least one nucleotide of the hybridized nucleic acid molecule using anucleotide polymerizing enzyme and a nucleotide (e.g., dATP, dTTP, dUTP,dCTP and/or a dGTP) or a nucleotide analog. Incorporation of anucleotide or a nucleotide analog is detected at discrete locations onthe surface. Nucleic acid target molecule/primer duplexes, as well asthe incorporated nucleotides, are individually resolvable in singlemolecule embodiments. Alternatively, bulk signal from mixed nucleic acidpopulations or clonal populations of nucleic acids, are obtained.

Fast reagent application and removal is another advantage of theinvention. For example, concentrations of nucleotides and/or otherreaction reagents can be alternated at different time points. This is aparticularly useful feature in an embodiment comprising introducing oneor more single species of nucleotide individually. This could lead toincreased incorporation rates and sensitivity. For example, when allfour types of nucleotides are simultaneously present in the reaction tomonitor dynamic incorporation of nucleotides, concentrations of the eachof the respective nucleotides can be alternated between a first and asecond range. This leads to both better visualization of the signal whenlow concentrations of nucleotides are present, and increasedpolymerization rate when higher concentrations of nucleotides arepresent.

The target nucleic acid can comprise or can be modified to comprise asequence that is sufficiently complementary to a primer to hybridize tothe primer to allow template dependent addition of nucleotides to thehybridized primer. The sequence complementary to the primer can be thesame as the previously described adaptor sequence. Therefore, theimmobilized capture probe and the primer can comprise the same sequence.The primer sequence can be about 10 to about 1000 nucleotides in length.The primer sequence and complementary target nucleic acid moleculesequence can be of the same length or of different lengths. Conditionsfor hybridizing primers to nucleic acid targets are well known. Theannealing reaction is performed under conditions which are stringentenough to guarantee sequence specificity, yet sufficiently permissive toallow formation of stable hybrids at an acceptable rate. The temperatureand length of time required for primer annealing depend upon severalfactors including the base composition, length and concentration of theprimer, and the nature of the solvent used, e.g., the concentration ofcosolvents such as DMSO (dimethylsulfoxide), formamide, or glycerol, andcounterions such as magnesium. Typically, hybridization (annealing)between primers and target nucleic acids is carried out at a temperaturethat is approximately 5 to 10° C. below the melting temperature of thetarget-primer hybrid in the annealing solvent. Typically, the annealingtemperature is in the range of 55 to 75° C. and the primer concentrationis approximately 0.2 μM. Under such conditions, the annealing reactionis usually complete within a few seconds.

Methods according to the invention include conducting a primer extensionreaction, such as exposing the nucleic acid/primer complexes topolymerase and one or more nucleotides under conditions sufficient toextend the primer by at least one base. Sequencing, as used herein canbe performed such that one or more nucleotides are identified in one ormore nucleic molecules. Methods according to the invention also includethe step of compiling a sequence of the molecule (nucleic acid) basedupon sequential incorporation of the extension bases into the primer.

In the analyzing step, the hybridized nucleic acid molecules can besequenced using single molecule sequencing as described, for example, inU.S. patent application Ser. No. 11/137,928, filed May 25, 2005 and/orand described in U.S. Pat. No. 6,780,591, the teachings of which areincorporated herein in their entirety. Polymerases useful in theinvention include any nucleic acid polymerase capable of catalyzing atemplate-dependent addition of a nucleotide or nucleotide analog to aprimer. Depending on the characteristics of the target nucleic acid, aDNA polymerase, an RNA polymerase, a reverse transcriptase, or a mutantor altered form of any of the foregoing can be used. According to oneaspect of the invention, a thermophilic polymerase is used, such asThermoSequenase®, 9°N™, Therminator™, Taq, Tne, Tma, Pfu, Tfl, Tth, Tli,Stoffel fragment, Vent™ and Deep Vent™ DNA polymerase.

The target nucleic acid molecule/primer complexes are contacted withdNTPs in the presence of the polymerase under conditions such that thepolymerase catalyzes template-dependent addition of a dNTP to the 3′terminus of the primer. The dNTP can be detectably labeled, as describedherein, and the nucleotide is identified by detecting the presence ofthe incorporated labeled nucleotide. As described above, unincorporatedlabeled dNTPs can be removed from the surface prior to detecting theincorporated labeled dNTP. The process can be repeated one or moretimes, wherein the template/primer complex(s) are provided withadditional dNTPs, in the presence of a polymerase, followed by removingthe unincorporated labeled dNTPs and detecting the incorporated labeleddNTP. The sequence of the template is determined by compiling thedetected (identified) dNTPs. In this manner, the entire sequence of oneor more templates can be determined. In addition, by using singlemolecule sequencing techniques, determining the sequence for eachnucleic acid molecule attached to the surface provides the number ofdifferent or unique nucleic acid molecules in the sample. Furthermore,the number of copies of each nucleic acid sequences in a biologicalsample is also provided.

In order to allow for further extension and detection of subsequentlyadded fluorophore-labeled nucleotides, the fluorophore of theincorporated nucleotide can be removed from the nucleotide incorporatedinto the primer. For example, the optical label (e.g., fluorescentlabel) can be destroyed by photochemical destruction as described inU.S. Pat. No. 6,780,591, the teachings of which are incorporated hereinin their entirety. This cycle can be repeated a large number of times ifsample losses are avoided. In one embodiment, such losses will beavoided by attaching the target nucleic acid molecules or primers to asurface of an array device, for example a microscope slide, andtransferring the entire array device between a reaction vessel and thefluorescent reader.

The extension reactions are carried out in buffer solutions whichcontain the appropriate concentrations of salts, dNTP(s) and nucleotidepolymerizing enzyme required for the enzyme mediated extension toproceed. For additional guidance regarding such conditions see, forexample, Sambrook et al., (1989, Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Press, NY); and Ausubel et al. (1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, NY).

Nucleotides particularly useful in the invention comprise detectablelabels. Labeled nucleotides include any nucleotide that has beenmodified to include a label that is directly or indirectly detectable.Preferred labels include optically-detectable labels, includingfluorescent labels or fluorophores, such as fluorescein, rhodamine,cyanine, cyanine-5 dye, cyanine-3 dye, or a derivative or modificationof any of the foregoing, and also include such labeling systems ashapten labeling. Accordingly, methods of the invention further providefor exposing the primer/target nucleic acid complex to a digoxigenin, afluorescein, an alkaline phosphatase or a peroxidase.

The sequencing can be optimized to achieve rapid and complete additionof the correct nucleotide to primers in primer/template complexes, whilelimiting the misincorporation of incorrect nucleotides. For example,dNTP concentrations may be lowered to reduce misincorporation ofincorrect nucleotides into the primer. K_(m) values for incorrect dNTPscan be as much as 1000-fold higher than for correct nucleotides,indicating that a reduction in dNTP concentrations can reduce the rateof misincorporation of nucleotides. Thus, in a preferred embodiment ofthe invention the concentration of dNTPs in the sequencing reactions areapproximately 5-20 μM.

In addition, relatively short reaction times can be used to reduce theprobability of misincorporation. For an incorporation rate approachingthe maximum rate of about 400 nucleotides per second, a reaction time ofapproximately 25 milliseconds will be sufficient to ensure extension of99.99% of primer strands.

Detection

Any detection method may be used that is suitable for the type of labelemployed. Thus, exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence orchemiluminescence. For example, extended primers can be detected on asubstrate by scanning all or portions of each substrate simultaneouslyor serially, depending on the scanning method used. For fluorescencelabeling, selected regions on a substrate may be serially scannedone-by-one or row-by-row using a fluorescence microscope apparatus, suchas described in Fodor (U.S. Pat. No. 5,445,934) and Mathies et al. (U.S.Pat. No. 5,091,652). Devices capable of sensing fluorescence from asingle molecule include scanning tunneling microscope (siM) and theatomic force microscope (AFM). Hybridization patterns may also bescanned using a CCD camera (e.g., Model TE/CCD512SF, PrincetonInstruments, Trenton, N.J.) with suitable optics (Ploem, in Fluorescentand Luminescent Probes for Biological Activity Mason, T. G. Ed.,Academic Press, Landon, pp. 1-11 (1993), such as described in Yershov etal., Proc. Natl. Aca. Sci. 93:4913 (1996), or may be imaged by TVmonitoring. For radioactive signals, a phosphorimager device can be used(Johnston et al., Electrophoresis, 13:566, 1990; Drmanac et al.,Electrophoresis, 13:566, 1992; 1993). Other commercial suppliers ofimaging instruments include General Scanning Inc., (Watertown, Mass. onthe World Wide Web at genscan.com), Genix Technologies (Waterloo,Ontario, Canada; on the World Wide Web at confocal.com), and AppliedPrecision Inc. Such detection methods are particularly useful to achievesimultaneous scanning of multiple attached target nucleic acids.

The present invention provides for detection of molecules from a singlenucleotide to a single target nucleic acid molecule. A number of methodsare available for this purpose. Methods for visualizing single moleculeswithin nucleic acids labeled with an intercalating dye include, forexample, fluorescence microscopy. For example, the fluorescent spectrumand lifetime of a single molecule excited-state can be measured.Standard detectors such as a photomultiplier tube or avalanchephotodiode can be used. Full field imaging with a two-stage imageintensified COD camera also can be used. Additionally, low noise cooledCCD can also be used to detect single fluorescent molecules.

The detection system for the signal may depend upon the labeling moietyused, which can be defined by the chemistry available. For opticalsignals, a combination of an optical fiber or charged couple device(CCD) can be used in the detection step. In those circumstances wherethe substrate is itself transparent to the radiation used, it ispossible to have an incident light beam pass through the substrate withthe detector located opposite the substrate from the target nucleicacid. For electromagnetic labeling moieties, various forms ofspectroscopy systems can be used. Various physical orientations for thedetection system are available and discussion of important designparameters is provided in the art.

A number of approaches can be used to detect incorporation offluorescently-labeled nucleotides into a single nucleic acid molecule.Optical setups include near-field scanning microscopy, far-fieldconfocal microscopy, wide-field epi-illumination, light scattering, darkfield microscopy, photoconversion, single and/or multiphoton excitation,spectral wavelength discrimination, fluorophore identification,evanescent wave illumination, and total internal reflection fluorescence(TIRF) microscopy. In general, certain methods involve detection oflaser-activated fluorescence using a microscope equipped with a camera.Suitable photon detection systems include, but are not limited to,photodiodes and intensified CCD cameras. For example, an intensifiedcharge couple device (ICCD) camera can be used. The use of an ICCDcamera to image individual fluorescent dye molecules in a fluid near asurface provides numerous advantages. For example, with an ICCD opticalsetup, it is possible to acquire a sequence of images (movies) offluorophores.

Some embodiments of the present invention use TIRF microscopy fortwo-dimensional imaging. TIRF microscopy uses totally internallyreflected excitation light and is well known in the art. See, e g., theWorld Wide Web at nikon-instruments.jp/eng/page/products/tirf.aspx. Incertain embodiments, detection is carried out using evanescent waveillumination and total internal reflection fluorescence microscopy. Anevanescent light field can be set up at the surface, for example, toimage fluorescently-labeled nucleic acid molecules. When a laser beam istotally reflected at the interface between a liquid and a solidsubstrate (e.g., a glass), the excitation light beam penetrates only ashort distance into the liquid. The optical field does not end abruptlyat the reflective interface, but its intensity falls off exponentiallywith distance. This surface electromagnetic field, called the“evanescent wave”, can selectively excite fluorescent molecules in theliquid near the interface. The thin evanescent optical field at theinterface provides low background and facilitates the detection ofsingle molecules with high signal-to-noise ratio at visible wavelengths.

The evanescent field also can image fluorescently-labeled nucleotidesupon their incorporation into the attached target nucleic acid targetmolecule/primer complex in the presence of a polymerase. Total internalreflectance fluorescence microscopy is then used to visualize theattached target nucleic acid target molecule/primer complex and/or theincorporated nucleotides with single molecule resolution.

Measured signals can be analyzed manually or by appropriate computermethods to tabulate results. The substrates and reaction conditions caninclude appropriate controls for verifying the integrity ofhybridization and extension conditions, and for providing standardcurves for quantification, if desired. For example, a control nucleicacid can be added to the sample. The absence of the expected extensionproduct is an indication that there is a defect with the sample or assaycomponents requiring correction.

Fluorescence resonance energy transfer (FRET) can be used as a detectionscheme. FRET in the context of sequencing is described generally inBraslavasky, et al., Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003),incorporated by reference herein. Essentially, in one embodiment, adonor fluorophore is attached to the primer, polymerase, or template.Nucleotides added for incorporation into the primer comprise an acceptorfluorophore that is activated by the donor when the two are inproximity.

As described herein, combination articles of manufacture are providedthat are suitable for performing the method of the invention. Suitablesurfaces are described above. The enzyme can be a ligase or apolymerase, as described above. The adaptor sequence can optionallycomprise a linker moiety at the 5′ terminus of the adaptor sequence, thelinker moiety being suitable for attaching a target molecule to thesurface. Other suitable linker moieties are described above.

EXAMPLE

The 7249 nucleotide genome of the bacteriophage M13mp18 was sequencedusing a single molecule system of the invention. Purified,single-stranded viral M13mp18 genomic DNA was obtained from New EnglandBiolabs. Approximately 25 ug of M13 DNA was digested to an averagefragment size of 40 by with 0.1 U Dnase I (New England Biolabs) for 10minutes at 37° C. Digested DNA fragment sizes were estimated by runningan aliquot of the digestion mixture on a precast denaturing (TBE-Urea)10% polyacrylamide gel (Novagen) and staining with SYBR Gold(Invitrogen/Molecular Probes). The DNase I-digested genomic DNA wasfiltered through a YM10 ultrafiltration spin column (Millipore) toremove small digestion products less than about 30 nt. Approximately 20pmol of the filtered DNase I digest was then polyadenylated withterminal transferase according to known methods (Roychoudhury, R and Wu,R. 1980, Terminal transferase-catalyzed addition of nucleotides to the3′ termini of DNA. Methods Enzymol. 65 (1):43-62.). The average dA taillength was 50+/−5 nucleotides. Terminal transferase was then used tolabel the fragments with Cy3-dUTP. Fragments were then terminated withdideoxyTTP (also added using terminal transferase). The resultingfragments were again filtered with a YM10 ultrafiltration spin column toremove free nucleotides and stored in ddH2O at −20° C.

Epoxide-coated glass slides were prepared for oligo attachment.Epoxide-functionalized 40 mm diameter #1.5 glass cover slips (slides)were obtained from Erie Scientific (Salem, N.H.). The slides werepreconditioned by soaking in 3×SSC for 15 minutes at 37° C. Next, a 500pM aliquot of 5′ aminated polydT(50) (polythymidine of 50 bp in lengthwith a 5′ terminal amine) was incubated with each slide for 30 minutesat room temperature in a volume of 80 ml. The resulting slides hadpoly(dT50) primer attached by direct amine linkage to the epoxide. Theslides were then treated with phosphate (1M) for 4 hours at roomtemperature in order to passivate the surface. Slides were then storedin polymerase rinse buffer (20 mM Tris, 100 mM NaCl, 0.001% TritonX-100, pH 8.0) until they were used for sequencing.

For sequencing, the slides were placed in a modified FCS2 flow cell(Bioptechs, Butler, Pa.) using a 50 um thick gasket. The flow cell wasplaced on a movable stage that is part of a high-efficiency fluorescenceimaging system built around a Nikon TE-2000 inverted microscope equippedwith a total internal reflection (TIR) objective. The slide was thenrinsed with HEPES buffer with 100 mM NaCl and equilibrated to atemperature of 50° C. An aliquot of the M13 template fragments describedabove was diluted in 3×SSC to a final concentration of 1.2 nM. A 100 ulaliquot was placed in the flow cell and incubated on the slide for 15minutes. After incubation, the flow cell was rinsed with1×SSC/HEPES/0.1% SDS followed by HEPES/NaCl. A passive vacuum apparatuswas used to pull fluid across the flow cell. The resulting slidecontained M13 template/oligo(dT) primer duplex. The temperature of theflow cell was then reduced to 37° C. for sequencing and the objectivewas brought into contact with the flow cell.

For sequencing, cytosine triphosphate, guanidine triphosphate, adeninetriphosphate, and uracil triphosphate, each having a cyanine-5 label (atthe 7-deaza position for ATP and GTP and at the C5 position for CTP andUTP (PerkinElmer)) were stored separately in buffer containing 20 mMTris-HCl, pH 8.8, 10 mM MgSO₄, 10 mM (NH₄)₂SO₄, 10 mM HCl, and 0.1%Triton X-100, and 100 U Klenow exo⁻ polymerase (NEN). Sequencingproceeded as follows.

First, initial imaging was used to determine the positions of duplex onthe epoxide surface. The Cy3 label attached to the M13 templates wasimaged by excitation using a laser tuned to 532 nm radiation (Verdi V-2Laser, Coherent, Inc., Santa Clara, Calif.) in order to establish duplexposition. For each slide only single fluorescent molecules were imagedin this step were counted. Imaging of incorporated nucleotides asdescribed below was accomplished by excitation of a cyanine-5 dye usinga 635 nm radiation laser (Coherent). 5 uM Cy5CTP was placed into theflow cell and exposed to the slide for 2 minutes. After incubation, theslide was rinsed in 1×SSC/15 mM HEPES/0.1% SDS/pH 7.0 (“SSC/HEPES/SDS”)(15 times in 60 ul volumes each, followed by 150 mM HEPES/150 mM NaCl/pH7.0 (“HEPES/NaCl”) (10 times at 60 ul volumes). An oxygen scavengercontaining 30% acetonitrile and scavenger buffer (134 ul HEPES/NaCl, 24ul 100 mM Trolox in MES, pH 6.1, 10 ul DABCO in MES, pH 6.1, 8 ul 2Mglucose, 20 ul NaI (50 mM stock in water), and 4 ul glucose oxidase) wasnext added. The slide was then imaged (500 frames) for 0.2 seconds usingan Inova301K laser (Coherent) at 647 nm, followed by green imaging witha Verdi V-2 laser (Coherent) at 532 nm for 2 seconds to confirm duplexposition. The positions having detectable fluorescence were recorded.After imaging, the flow cell was rinsed 5 times each with SSC/HEPES/SDS(60 ul) and HEPES/NaCl (60 ul). Next, the cyanine-5 label was cleavedoff incorporated CTP by introduction into the flow cell of 50 mM TCEPfor 5 minutes, after which the flow cell was rinsed 5 times each withSSC/HEPES/SDS (60 ul) and HEPES/NaCl (60 ul). The remaining nucleotidewas capped with 50 mM iodoacetamide for 5 minutes followed by rinsing 5times each with SSC/HEPES/SDS (60 ul) and HEPES/NaCl (60 ul). Thescavenger was applied again in the manner described above, and the slidewas again imaged to determine the effectiveness of the cleave/cap stepsand to identify non-incorporated fluorescent objects.

The procedure described above was then conducted 100 nM Cy5dATP,followed by 100 nM Cy5dGTP, and finally 500 nM Cy5dUTP. The procedure(expose to nucleotide, polymerase, rinse, scavenger, image, rinse,cleave, rinse, cap, rinse, scavenger, final image) was repeated exactlyas described for ATP, GTP, and UTP except that Cy5dUTP was incubated for5 minutes instead of 2 minutes. Uridine was used instead of thymidinedue to the fact that the Cy5 label was incorporated at the positionnormally occupied by the methyl group in Thymidine triphosphate, thusturning the dTTP into dUTP. In all 64 cycles (C, A, G, U) were conductedas described in this and the preceding paragraph.

Once 64 cycles were completed, the image stack data (i.e., the singlemolecule sequences obtained from the various surface-bound duplex) werealigned to the M13 reference sequence. The image data obtained wascompressed to collapse homopolymeric regions. Thus, the sequence“TCAAAGC” would be represented as “TCAGC” in the data tags used foralignment. Similarly, homopolymeric regions in the reference sequencewere collapsed for alignment. The sequencing protocol described aboveresulted in an aligned M13 sequence with an accuracy of between 98.8%and 99.96% (depending on depth of coverage). The individual singlemolecule sequence read lengths obtained ranged from 2 to 33 consecutivenucleotides with about 12.6 consecutive nucleotides being the averagelength.

The alignment algorithm matched sequences obtained as described abovewith the actual M13 linear sequence. Placement of obtained sequence onM13 was based upon the best match between the obtained sequence and aportion of M13 of the same length, taking into consideration 0, 1, or 2possible errors. All obtained 9-mers with 0 errors (meaning that theyexactly matched a 9-mer in the M13 reference sequence) were firstaligned with M13. Then 10-, 11-, and 12-mers with 0 or 1 error werealigned. Finally, all 13-mers or greater with 0, 1, or 2 errors werealigned. At a coverage depth of greater than or equal to 1, 5,001 basesof the 5,066 base M13 collapsed genome were covered at an accuracy of98.8%. Similarly, at a coverage depth of greater than or equal to 5,83.6% of the genome was covered at an accuracy of 99.3%, and at a depthof greater than or equal to 10, 51.9% of the genome was covered at anaccuracy of 99.96%. The average coverage depth was 12.6 nucleotides.

1-25. (canceled)
 26. A method for obtaining sequence information from anucleic acid, the method comprising the steps of: introducing a samplecomprising at least one target nucleic acid and other non-targetmolecules to at least one sequence-specific capture probe to producetarget/probe duplexes; separating at least one of the duplexes from thenon-target molecules; exposing the separated duplexes to at least onelabeled nucleotide in the presence of a polymerase capable of catalyzingaddition of the nucleotide to the duplex in a template-dependent manner,wherein the probe acts as a primer; and detecting incorporation of thenucleotide into the probe portion, thereby obtaining sequenceinformation from the nucleic acid.
 27. The method according to claim 26,wherein prior to the exposing step, the duplexes are attached to asurface.
 28. The method according to claim 27, wherein the duplexes aredirectly attached to a surface.
 29. The method according to claim 27,wherein the duplexes are indirectly attached to a surface.
 30. Themethod according to claim 27, wherein at least some of the attachedduplexes are individually optically resolvable.
 31. The method accordingto claim 26, wherein the probes are a plurality of differentsequence-specific probes.
 32. The method according to claim 26, whereinthe exposing and detecting steps are repeated at least once.
 33. Themethod according to claim 32, further comprising: removingunincorporated nucleotide and polymerase in all or some repetitions ofthe exposing and detecting steps.
 34. The method according to claim 26,wherein the labeled nucleotide is a detectably labeled nucleotide. 35.The method according to claim 34, wherein the detectably labelednucleotide is an optically detectably labeled nucleotide.
 36. The methodaccording to claim 35, wherein the optically detectably labelednucleotide is a fluorescently labeled nucleotide.
 37. The methodaccording to claim 26, wherein the target nucleic acid comprises atleast one mutation indicative of a disease state.
 38. The methodaccording to claim 26, wherein prior to the introducing step, the methodfurther comprising modifying the target nucleic acid with an adaptorsequence.
 39. The method according to claim 38, wherein each targetnucleic acid is modified with a different adaptor sequence.
 40. A methodfor obtaining sequence information from a nucleic acid, the methodcomprising the steps of: contacting a sample comprising at least onetarget nucleic acid and other non-target molecules to at least onesequence-specific capture probe attached to a surface to producetarget/probe duplexes; separating at least one of the duplexes from thenon-target molecules; exposing the separated duplexes to at least onelabeled nucleotide in the presence of a polymerase capable of catalyzingaddition of the nucleotide to the duplex in a template-dependent manner,wherein the probe acts as a primer; and detecting incorporation of thenucleotide into the probe portion, thereby obtaining sequenceinformation from the nucleic acid.
 41. The method according to claim 40,wherein at least some of the attached duplexes are individuallyoptically resolvable.
 42. The method according to claim 40, wherein theexposing and detecting steps are repeated at least once.
 43. The methodaccording to claim 42, further comprising: removing unincorporatednucleotide and polymerase in all or some repetitions of the exposing anddetecting steps.
 44. The method according to claim 40, wherein theprobes are directly attached to the surface.
 45. The method according toclaim 40, wherein the probes are indirectly attached to the surface. 46.The method according to claim 40, wherein the probes are a plurality ofdifferent sequence-specific probes.
 47. A method for obtaining sequenceinformation from a nucleic acid, the method comprising the steps of:performing hybrid capture on a sample comprising at least one targetnucleic acid and other non-target molecules to produce target/probeduplexes; separating at least one of the duplexes from the non-targetmolecules; and performing a sequencing by synthesis reaction on theseparated target/probe duplexes, wherein the probe acts as a primer forthe sequencing reaction.